Seroprevalence of Toxoplasma gondii in Pregnant Women and Bioassay of IgM Positive Cases in Zanjan, Northwest of Iran

Background The aim of this study was to determine the Toxoplasma antibodies in pregnant women in Zanjan, by ELISA method. Methods Blood samples were taken from 500 pregnant women referred to the health centers of Zanjan City, North West Iran, IgM and IgG titers were primarily evaluated. The collected data were analyzed with SPSS 11.5 using Chi-Square test. Results Anti Toxoplasma IgM and IgG were positive in 1.4% and 37.2% respectively. Seropositive subjects were more frequently seen in those with >30 years old compared to younger women (<20 years old). No significant relationship was found between the seroprevalence of T. gondii infection and level of education, residence area, history of abortion and gestational age. Conclusion The rate of IgM positive was low; however, a large number of the studied population were IgG positive, indicative of having a latent infection due to the past exposure to Toxoplasma parasite in this region.


Introduction
oxoplasmosis is a worldwide zoonosis that can infect approximately one third of the world's population by a protozoan parasite, Toxoplasma gondii. However, it may appear in several forms, infections in humans are usually clinically unapparent, and latent infection usually persists for life, which is not seen as a health threat except in case of pregnancy and immunocompromised ind-ividuals. Toxoplasma gondii can be transmitted to humans by three principal ways; first, by eating raw or uncooked meat, especially pork, mutton or uncooked food and those who are in contact with infected meat. Second, by consuming oocysts those cats have passed in their feces, either from a trash box or from soil (e.g., soil from gardening, or unwashed fruit or vegetables, or in unrefined water). Third, by transmitting the infection from infected pregnant women to their unborn fetus it can be happened. It is known that the parasite can cause congenital disease and abortion both in humans and livestock. In most cases the laboratory diagnosis of acute and latent toxoplasmosis relies on the detection of T. gondii specific IgG and IgM antibodies and the avidity test of T. gondii specific IgG antibodies has also been very helpful in the diagnosis. Many serological tests such as the latex agglutination test, ELISA, indirect fluorescence antibody test (IFA) and hemagglutination test have been used in the detection of antibodies against T. gondii in pregnant women (1,2). There are many studies on the prevalence of anti-T. gondii antibody among Iranian women. Seropositivity of T. gondii is 48%-74.6% in northern areas (3)(4)(5), 33%-44% in northwest (6-10), 22%-37% in south (11)(12)(13) and 27%-54% in central parts of Iran (14)(15)(16)(17). The seroprevalence of the infection in pregnant women in Zanjan was reported as 17.9% (18,19). The present study was performed to determine the Toxoplasma antibodies in pregnant women in Zanjan, by ELISA method because of its high sensitivity and specificity, easier technique and lower expanse which is preferred in order to screening Toxoplasma infection.

Materials and Methods
This study was carried out in Zanjan City, North West Iran; which has an area of 36,400 km². Zanjan has a highland climate characterized by cold snowy weather in the mountains and moderate climate in the plains in winter time. In the summer, the weather is warm. The average maximum temperature of Zanjan is around 27 °C, whereas the average minimum temperature stands at -19 °C.

Patients and blood sampling
Three ml of venous blood sample were drowning from the study group (500 pregnant women) and anticuagulated with citrated sodium. Two ml of citrated blood were centrifuged at 3000 rpm for 15 minutes. The plasma was isolated and stored at 4 °C until the assays. Serum samples were tested for anti Toxoplasma IgM and IgG antibodies using Toxoplasma IgM ELISA kit, GD81 and Toxoplasma IgG ELISA kit, GD80 (Genesis Diagnostic Company/England). The present work has been approved by Zanjan University of Medical Sciences Ethics Committee.

Statistical analysis
The chi-square test was used to analyze the data in SPSS version 11.5.

Results
The overall seroprevalence of toxoplasmosis in pregnant women was 37.8% (189/500 cases). IgG and IgM anti-Toxoplasma antibodies were positive in 186/500 cases (37.2%) and 7/500 cases (1.4%), respectively. The results, T including seroprevalence data together with personal and demographic variables are detailed in Table 1. The proportion of seropositive women increased with increasing age, from 28.7% in ≤20 years old to 48% in >30 years old women (P= 0.025). No significant relationship was found between the seroprevalence of T. gondii infection and their level of education. The prevalence rate showed no sig-nificant differences between women resident in rural and those in urban areas, neither the history of abortion had significant association with Toxoplasma seroprevalence rate. The surveyed pregnant women at their first, second and third gestational trimesters showed indifferent rates of the Toxoplasma infection. The data of the above variables are summarized in Table 1.

Discussion
A high prevalence of chronic toxoplasmosis (IgG positive) during pregnancy was found in this study (37.2%) that is accordance with weather and geological conditions of this area. In Iran, at least 30% of people have been found seropositive for anti T. gondii immunoglobulin G (IgG) in most regions (7). A seroprevalence of 17.9% in pregnant women referred to a hospital in Zanjan was reported (18) which was lower than what we found. The authors used indirect fluorescent antibody test (IFT), whereas we used ELISA kit; of course, that much difference between these two studies cannot be solely attributed to the different serological methods. It is noteworthy that the interval between these two studies is more than a decade. Further, the referral hospitals of the two studies were different in several aspects. In our study, the prevalence of recently acquired infections (IgM positive) was relatively low (1.4%). The IgG seropositive rate increased with age. Women older than 30 years had a significantly higher seroprevalence (48%) compared to those who were 20 or less (28.7%) (P= 0.025). Low level of education was associated with higher rate of toxoplasmosis (18)(19)(20)(21). We did not find a significant relationship between the seroprevalence of T. gondii infection and the level of education. There are similar reports in Turkey and Hamadan (6,22). Some other studies showed a significance decreases in seropositivity as the level of the education increases (7,13,23). In the present study, we found no statistical difference among seroprevalence of T. gondii and the residence areas, which is in accordance with other reports (7,22,24), but some studies showed higher seropositivity in urban than in rural areas (25,26). In addition, in the present research similar to the results of previous studies, (18,22), no significant relationship was found between the seroprevalence of T. gondii infection and history of abortion in pregnant women. No significant association of the infection with gestational age was observed.
In conclusion, this study showed that exposure to T. gondii parasite in the past is considerable in the pregnant women in Zanjan and at the same time many of them remain susceptible to the infection and require care to avoid parasite exposure during their pregnancies.